Charakterisierung von frühen Komponenten der Signaltransduktion in Ustilago maydis

In U. maydis pathogenic development is initiated by fusion of two compatible strains. Fusion is regulated by a pheromone-receptor system, and pheromone signaling involves the activation of a conserved cAMP pathway as well as of a MAP kinase module. Heterotrimeric G-proteins are considered as the initial transmitters of the pheromone signal during mating partner recognition in filamentous fungi and yeasts. U. maydis encodes four Ga-subunits gpa1-4, but only to Gpa3 a function in the cAMP pathway could be assigned. It remained unclear if the three remaining Ga-subunits are functional redundant between each other or to Gpa3. In this study, the C-terminus of the pheromone receptor Pra1 could be identified as functional important site for signal transduction. Cells expressing a C-terminal truncated receptor protein are unable to form conjugation hyphae upon pheromone stimulation but remain competent to fuse with wild type cells and to establish dikaryotic hyphae. This defect could be ascribed to the lack of MAP kinase module activation and indicates interaction of an activating component with the Pra1-C-terminus. To test for redundancy in Ga-subunit function, strains deleted in gpa1, gpa2, gpa4 and strains deleted in all Ga-subunits were generated. gpa1 gpa2 gpa4-triple mutants were indistinguishable from wild type in their pheromone response and pathogenic development. However, spore formation was reduced in these strains. Therefore, the Ga-subunits gpa1 gpa2 gpa4 are not essential for completion of the U. maydis life cycle. Ga null mutants were phenotypically indistinguishable from gpa3-mutants and could be reverted by external cAMP. These results argue against functional redundancy between Ga-subunits and suggest that only Gpa3 is needed for pheromone signaling. The suppression of phenotypes by cAMP could be extended to appressoria formation and penetration. The induction of "budding filaments" in these strains on hydrophobic surfaces including the leaf surface could be described for the first time. However, full pathogenicity could not be achieved by cAMP supplementation indicating essential functions of Gpa3 during tumour development. Gpa3 represents therefore the major regulatory Ga-subunit for growth and pathogenic development in U. maydis. Microarray analyses revealed in the triple mutant few genes with lower basal expression compared to wild type. These genes encoded proteins with functions in the pheromone response and metabolism; however these differences did not affect the pheromone response in this strain. The Analysis of the gpa3 mutant transcriptome led to the identification of 163 differentially expressed genes with functions in metabolism, nutrient transport and morphology. This is consistent with the role of Gpa3 in regulating the cAMP pathway. Therefore, signaling through Gpa3 reflects the nutrient status of the cell and participates in glucose repression of genes. The clear differences to the transcriptome of the triple mutant confirm the functional non-redundant role of Gpa3 as a central activator of the cAMP pathway.